Roberta Pena da Paschoa^1,2, Lucas Rodrigues Xavier^1,2, Caio Cezar Guedes Corrêa^1,2, Karina da Silva Vieira^1,2, Daniel Dastan Rezabala Pacheco¹, Lucas do Espirito Santo Gomes³, Carlos Eduardo Assis da Silva^1,2, Laís dos Santos da Conceição^1,2, Vitor Batista Pinto³, Claudete Santa-Catarina³, Vanildo Silveira^1,2

Sugarcane (Saccharum spp. hybrid) is an economically important crop widely cultivated for its high sucrose content. Somatic embryogenesis represents a promising strategy for efficient micropropagation and genetic transformation in this crop. The induction of somatic embryogenesis is regulated by multiple genes and proteins associated with hormonal signaling and stress responses, which act as key regulators of cellular dedifferentiation and differentiation under in vitro conditions. In this study, we employed a temporal proteomic approach combined with global DNA methylation (GDM) analysis to investigate the molecular mechanisms underlying sugarcane embryogenic callus induction in response to 2,4-dichlorophenoxyacetic acid (2,4-D). Proteomic profiling revealed 1,011 differentially accumulated proteins (DAPs) across at least one of the evaluated time point comparisons (0, 7, 14, and 21 days). These DAPs were grouped based on their accumulation patterns, revealing four main clusters that highlighted proteins associated with embryogenesis, epigenetic regulation, hormonal signaling, and post-translational modifications. Proteins related to photosynthesis and starch accumulation were downregulated during callus induction, indicating a metabolic reprogramming associated with cellular differentiation. In contrast, proteins linked to embryogenic competence, epigenetic regulation, hormonal responses, and post-translational control were enriched during the induction period. Among the predicted regulatory interactions, we identified TOPLESS, SKP1, CUL1, CAND1, and the histone deacetylase HDT2, suggesting auxin-mediated transcriptional repression coupled with chromatin remodeling. Several phosphatases (PP2A-1, PP2A-2, PP1) also accumulated, supporting their involvement in hormonal responses and cell cycle control. Additionally, epigenetic regulators such as adenosylhomocysteinase, SUVH1-like, and Argonaute proteins were identified, potentially contributing to the acquisition of embryogenic competence. GDM levels ranged from 13.56% to 16.46% throughout the induction period, indicating that epigenetic modulation may play a significant role in embryogenic competence acquisition. Altogether, our findings indicate that embryogenic callus induction in sugarcane is driven by a complex regulatory network involving photosynthetic repression, hormonal signaling, epigenetic remodeling, and tight control of translation and the cell cycle. This study advances our understanding of the molecular basis of somatic embryogenesis in sugarcane and highlights key regulatory proteins that may be targeted to improve somatic embryogenesis protocols and biotechnological applications in this crop.

Agradecimentos: Universidade Estadual do Norte Fluminense Darcy Ribeiro; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).